Controlling Monoterpene Isomerization by Guiding Challenging Carbocation Rearrangement Reactions in Engineered Squalene-Hopene Cyclases.

The interconversion of monoterpenes is facilitated by a complex network of carbocation rearrangement pathways. Controlling these isomerization pathways is challenging when using common Brønsted and Lewis acid catalysts, which often produce product mixtures that are difficult to separate. In contrast, natural monoterpene cyclases exhibit high control over the carbocation rearrangement reactions but are reliant on phosphorylated substrates. In this study, we present engineered squalene-hopene cyclases from Alicyclobacillus acidocaldarius (AacSHC) that catalyze the challenging isomerization of monoterpenes with unprecedented precision. Starting from a promiscuous isomerization of (+)-ß-pinene, we first demonstrate noticeable shifts in the product distribution solely by introducing single point mutations. Furthermore, we showcase the tuneable cation steering by enhancing (+)-borneol selectivity from 1 % to >90 % (>99 % de) aided by iterative saturation mutagenesis. Our combined experimental and computational data suggest that the reorganization of key aromatic residues leads to the restructuring of the water network that facilitates the selective termination of the secondary isobornyl cation. This work expands our mechanistic understanding of carbocation rearrangements and sets the stage for target-oriented skeletal reorganization of broadly abundant terpenes.

An enhanced toluene dioxygenase platform for the production of cis-1,2-dihydrocatechol in Escherichia coli BW25113 lacking glycerol dehydrogenase activity.

The compound cis-1,2-dihydrocatechol (DHC) is highly valuable since it finds wide application in the production of fine chemicals and bioactive compounds with medical relevance. The biotechnological process to generate DHC involves a dearomatizing dihydroxylation reaction catalyzed by toluene dioxygenase (TDO) from P. putida F1, employing benzene as substrate. We aimed to enhance the biotechnological E. coli BW25113 platform for DHC production by identifying the key operational parameters positively influencing the final isolated yield. Thereby, we observed an unreported downstream reaction, generating catechol from DHC, affecting, in a negative manner, the final titer for the product. Expression temperature for the TDO-system showed to have the highest influence in terms of final isolated yield. A KEIO-collection-based screening approach highlighted glycerol dehydrogenase (GldA) as the main responsible enzyme for the undesired reaction. We transferred the TDO-system to E. coli BW25113 ΔgldA and applied the enhanced operational set-up on it. This enhanced platform enabled the production of 1.41 g L-1 DHC in isolated yield, which represents a two-fold increase compared with the starting working conditions. To our knowledge, this is the highest DHC production accomplished in recombinant E. coli at semi-preparative scale, providing a robust and accessible biotechnological platform for DHC synthesis.

Strategy for identification of cis-dihydrodiendiol-degrading dehydrogenases in E. coli BW25113.

cis-Dihydrodiendiols are valuable compounds, finding multiple application as chiral synthons in organic chemistry. The biotechnological route for the generation of cis-dihydrodiendiols involves the dihydroxylation of aromatic compounds, catalyzed by Rieske non-heme iron dioxygenases. To date, numerous examples of recombinant E. coli, harboring such dioxygenases, can be found in the literature. Nevertheless, there is only a minor number of publications, addressing the E. coli catalyzed degradation of cis-dihydrodiendiols into catechols via dehydrogenases. Identification and elimination of such dehydrogenase catalyzed degradation is key for the establishment of enhanced recombinant E. coli platforms pursuing the production of cis-dihydrodiendiols. Here, we provide a fast and easy strategy for the identification of promiscuous alcohol dehydrogenases in E. coli BW25113, catalyzing the degradation of cis-dihydrodiendiols into catechols. This approach is based on the screening of dehydrogenase deficient KEIO strains, regarding their incapability of degrading a cis-dihydrodiendiol of choice.•Novel screening strategy for E. coli BW25113 dehydrogenase knock-outs, incapable of degrading cis-dihydrodiendiols was validated for cis-1,2-dihydrocatechol as substrate•Corresponding knock-outs can be used for recombinant production of cis-dihydrodiendiols•Simple analysis based on liquid chromatography with diode array detector (HPLC-DAD).

Quantifying ribosome dynamics in Escherichia coli using fluorescence.

Ribosomes are a crucial component of the physiological state of a cell. Therefore, we aimed to monitor ribosome dynamics using a fast and easy fluorescence readout. Using fluorescent-labeled ribosomal proteins, the dynamics of ribosomes during batch cultivation and during nutritional shift conditions was investigated. The fluorescence readout was compared to the cellular rRNA content determined by capillary gel electrophoresis with laser-induced fluorescence detection during exponentially accelerating and decelerating growth. We found a linear correlation between the observed fluorescence and the extracted rRNA content throughout cultivation, demonstrating the applicability of this method. Moreover, the results show that ribosome dynamics, as a result of slowing growth, are accompanied by the passive effect of dilution of preexisting ribosomes, de novo ribosome synthesis and ribosome degradation. In light of the challenging task of deciphering ribosome regulatory mechanisms, our approach of using fluorescence to follow ribosome dynamics will allow more comprehensive studies of biological systems.